Severe congenital T lymphopenia occurs in DiGeorge anomaly as a result of a developmental defect of the thymus gland. Surgical implantation of postnatal, allogeneic, cultured thymic fragments has been performed in an attempt to provide an appropriate microenvironment for thymopoiesis in these patients. Despite some success with this approach, peripheral T cell numbers have remained consistently low and autoimmune disease is a frequent problem. Detailed analysis of the cellular mechanisms of thymic reconstitution that occur within the implants is not possible in the clinical setting. Our goal in this proposal is to use a human marrow- thymus chimera in an immune deficient mouse model to create a platform from which we can delineate on a cellular level how thymocytes are generated in the human thymic implants, and how thymopoiesis might be improved. To achieve this goal, we will bring together information from our studies of normal human thymopoiesis, and of the thymic vascular niche. Using immune deficient murine models, we have noted that the neonatal thymus provides a uniquely receptive environment for rapid thymic seeding and thymopoiesis after hematopoietic stem cell transplantation. Our data shows that qualitative differences exist between the neonate and adult thymic vasculature, and that these differences are mediated by high levels of Vascular Endothelial Growth Factor (VEGF). We hypothesize that the VEGF responsive vasculature of the neonatal thymus mediates rapid and robust thymopoiesis. Furthermore, we propose that expression of VEGF in implanted thymic tissue will improve the speed and quality of thymic reconstitution from host hematopoietic cells. We propose the following Specific Aims: 1. To determine the cellular mechanisms by which reconstitution of human thymopoiesis is established after implantation of postnatal cultured thymus. 2. To determine if VEGF expression in human postnatal thymic implants will improve implant survival and thymopoiesis. In addition to the direct relevance of these studies to the treatment of DiGeorge anomaly, these studies will provide a technically feasible approach to manipulate the human thymic microenvironment experimentally ex vivo, and examine the biology of such manipulations in the context of endogenous human hematopoiesis. Understanding the role of the vascular niche in thymic reconstitution may provide novel insight into mechanisms of cross-talk within the cellular compartments of the thymus. PUBLIC HEALTH RELEVANCE: The ability to target expression of molecules specifically to the human thymus has broad therapeutic potential for both primary immune deficiencies like DiGeorge anomaly and for acquired states of thymic insufficiency that develop throughout postnatal life.